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High doses of anabolic androgenic steroid are associated with changes in personality, e.g. increased aggression and irritability, behavioural changes that may be linked to structural changes in the hippocampus. In this in vivo study we demonstrate acute effects of a single injection of 19-nortestosterone on proteins that play a major role in molecular plasticity at synaptic connections. The steroid rapidly and transiently decreased total and phosphorylated NMDA receptor GluN2B subunit levels and phosphorylated extracellular signal-regulated kinase 1 in rat hippocampal synaptoneurosomes. Pretreatment with the androgen receptor antagonist flutamide prevented these effects suggesting an androgen receptor mediated mode of action. However, flutamide alone stimulated the phosphorylation of both extracellular signal-regulated kinase 1 and 2. EphrinB2 and phosphorylated translation initiation factor 4E, two proteins that act on synaptic plasticity through NMDA receptor and/or mitogen-activated protein kinase pathways, were not affected by any of the treatment regimens. This study demonstrates rapid in vivo effects of an anabolic androgenic steroid on two key elements in hippocampal synaptic plasticity.

Neurosteroids are endogenously derived compounds, mediating rapid effects in the central nervous system. They participate in vital processes, including memory and learning, neuroplasticity, and neuroprotection in Alzheimer's disease. However, the mechanisms behind those effects remain to be elucidated. The neurosteroids pregnenolone sulphate (PS) and pregnanolone sulphate (3alpha5betaS) have recently been shown to allosterically alter the NMDA receptor in nanomolar concentrations. Those studies featured ifenprodil, which is a dirty drug, with affinity to many targets. In this study we compare the NMDA receptors in the hippocampus to recombinant NMDA receptors, using [3H]-MK-801 as radioligand. The results show that neurosteroids modulate the ifenprodil binding kinetics in a narrow concentration interval, addressing it to the NR2B subunit, since no effects were recorded at recombinant NR1/NR2A receptors. The effects were also seen as changes in the manner ifenprodil displaced or induced the dissociation of [3H]-MK-801. It indicates that the neurosteroidal effects indeed alter the ion pore of the NMDA receptor, why it is reasonable to believe that these findings have physiological relevance.

The age-related decline in gonadal steroids is associated with changes in mood and memory function. It appears that normal physiological concentrations of the steroids are required for adequate synaptic plasticity. However, the effects of high levels of androgens subsequent to misuse of anabolic androgenic steroids (AAS) are largely unknown. In this study, rats were given i.m. nandrolone as a single dose or daily for 14 days and the effects on synaptic components in hippocampal synaptoneurosomes were measured 24h after the last injection. Western blot analysis revealed that a single injection of AAS increased phosphorylation of the NMDA receptor subunits NR2A and NR2B and ERK1/2, while the levels of phosphorylated CaMKIIalpha were unaltered. No changes were seen in other synaptic proteins tested, i.e., BDNF, Arc, TUC-4, and beta-tubulin III. Daily administration of nandrolone for 2 weeks did not affect the content of any of the proteins tested. From this in vivo study, it is concluded that important synaptic components respond to a single high dose of nandrolone, an effect that may influence synapse function.

Insulin-like growth factor-1 (IGF-1) and growth hormone (GH) have been suggested to promote memory and cognitive capabilities. In a recent publication we observed that GH increase the proportion of the NR2B subunit mRNA transcript of the NMDA receptor in rat hippocampus. NR2B has been suggested to be essential for spatial learning and long-term potentiation (LTP). This effect of GH might be IGF-1-mediated or a result of a co-ordination with IGF-1. To test this hypothesis further, we examined the effects of 10 daily s.c. injections of IGF-1 on NMDA receptor subunits (NR1, NR2A, and NR2B), GH receptor (GHR), GH binding protein (GHBP) and type 1 IGF receptor (IGF-1R) gene transcripts in the hippocampus. The NR2B subunit mRNA increased in young (11 weeks) but not in older (14-16 months) rats and the expression of the NR2A mRNA was decreased in both groups. The ratio of NR2B to NR2A is suggested to mirror the potential for synaptic plasticity. In both age groups, IGF-1 treatment resulted in a significant increase of this ratio at transcription level. The GHR mRNA increased in young rats, mimicking the effect of GH, while the IGF-1R mRNA was decreased in the older group of rats after IGF-1 treatment. These results suggest that IGF-1 in many aspects may mediate the actions earlier shown for GH.

In the past, several studies have been conducted investigating the impact of androgens on the activation of known signaling pathways in cultured primary hippocampal cells and C6 cells. We have recently reported two studies showing altered in vivo phosphorylation of key components in cellular signaling in rat hippocampal synaptoneurosomes after administration of anabolic androgenic steroids. In this paper we report a quantitative liquid chromatography mass spectrometry-based screening for androgen-induced alterations of phosphoproteins in rat hippocampal synaptoneurosomes following a single 19-nortestosterone administration. We identified over 80 phosphorylated peptides with functions associated with the cytoskeleton and transmembrane signaling. DeCyder™ MS based quantitative comparison of the relative phosphopeptide abundances between the two groups of treatment was computed for the majority of the phosphopeptides.